Neural crest cells serve as progenitors for many important cell types, including neurons and glia of the peripheral nervous system and cartilage of the craniofacial skeleton. Elegant embryological studies have revealed key insights into the tissue and cellular interactions that control neural, crest cell specification ,migration and differentiation. Little is known, however, about the genetic mechanisms that control these processes. The goal of this proposal is to define molecular mechanisms controlling specification and differentiation of cranial neural crest cells in the developing vertebrate nervous system and craniofacial skeleton. Zebrafish will be used primarily for these studies since it is an advantageous system for studies involving both genetics and embryolqgy. I have recently determined that novel cell autonomous zebrafish mutation, narrowminded (nrd), is an essential regulator for the development of cranial neural crest cells. This proposal aims to molecularly characterize nrd, which will serve as a starting point in the isolation of additional regulators of the neural crest. Positional cloning of nrd, and the identification of candidate genes for he mutation has been initiated, both with the end goal of cloning the gene. Once the gene has been cloned, the further characterization the nrd gene product will be initiated in cranial neural crest cell patterning. This will include the analysis of the craniofacial phenotype of nrd by expression analysis, along with perturbation of endogenous nrd expression. To understand how nrd affects cell fate, lineage analysis will be done to correlate cell fate with morphological structure and localize patterns of gene expression. The results will lay the foundation for future experiments involving cranial neural crest cells. Finally, aim 3 will evaluate the role of other cell autonomous factors, such as Dlx3, that function downstream of nrd in neural crest cell formation and differentiation. Preliminary data using dominant negative and active mutants of Dlx3 suggest that it is crucial for correct patterning of the neural/non-neural ectodermal border region, from where neural crest, placodal cells and sensory neurons originate. The further characterization of nrd and downstream genes involved in cranial neural crest cell development will add to our understanding of this highly migratory population with the hope of preventing congenital disorders within craniofacial and other neural crest derived tissues, such as in Pierre Robin and DiGeorge syndromes.